Komesuamide and odopenicillatamide, two linear lipopeptides from the marine cyanobacterium Caldora penicillata

Article abstract of DOI:10.1016/j.tet.2021.131969

The linear lipopeptides komesuamide (1) and odopenicillatamide (2) were isolated from Caldora penicillata a marine cyanobacterium collected in Okinawa. The structures of these compounds were established by spectroscopic analyses, and the absolute configurations were determined by HPLC analyses of the acid hydrolysates. Both compounds showed glucose uptake activity at 40 μM in cultured L6 myotubes.

Full text of DOI:10.1016/j.tet.2021.131969

Tetrahedron 85 (2021) 131969
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Tetrahedron
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Komesuamide and odopenicillatamide, two linear lipopeptides from
the marine cyanobacterium Caldora penicillata
Kaori Ozaki a, Atsuhide Jinno a, Noriyuki Natsume a, Shimpei Sumimoto b,
, d, *
Arihiro Iwasaki c, Kiyotake Suenaga c, Toshiaki Teruya a
a Graduate School of Engineering and Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, 903-0213, Japan
b Department of Material and Life Chemistry, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama, 221-8686, Japan
c Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa, 223-8522, Japan
d Faculty of Education, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, 903-0213, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The linear lipopeptides komesuamide (1) and odopenicillatamide (2) were isolated from Caldora pen-
icillata a marine cyanobacterium collected in Okinawa. The structures of these compounds were
established by spectroscopic analyses, and the absolute configurations were determined by HPLC ana-
Received 11 November 2020
Received in revised form
21 January 2021
Accepted 23 January 2021
Available online 30 January 2021
lyses of the acid hydrolysates. Both compounds showed glucose uptake activity at 40
myotubes.
mM in cultured L6
© 2021 Elsevier Ltd. All rights reserved.
Keywords:
Linear lipopeptide
Glucose uptake activity
Marine cyanobacteria
Caldora penicillata
1. Introduction
against HeLa cells, inhibitory activity against sterol O-acyl-
transferase, and antimalarial activity, respectively. Meanwhile, we
recently reported that the linear lipopeptides ypaoamide B and
mabuniamide showed glucose uptake activity in cultured L6
myotubes [11,12]. This suggested that marine cyanobacteria may be
a source of compounds with glucose uptake regulatory properties.
Accordingly, we searched for constituents of marine cyanobac-
teria whose crude organic extracts showed glucose uptake activity
in L6 myotubes, resulting in the isolation of komesuamide (1) and
odopenicillatamide (2) (Fig. 1). Here, we report the isolation,
structure elucidation, and biological activities of 1 and 2.
Diabetes mellitus (DM) is a major health problem in the modern
world. In 2014, a reported 422 million people were living with DM,
which causes 1.6 million deaths annually [1]. Type 2 DM (T2DM),
characterized by insulin resistance and hyperglycemia, accounts for
over 90% of DM cases globally [2]. Because T2DM can lead to
complications such as nephropathy, neuropathy, and retinopathy
[3], its spread may impair patients’ quality of life and impose an
economic burden on health care systems. There is thus an urgent
need for the development of therapeutic drugs for the treatment of
T2DM.
In the field of drug discovery, marine organisms are a rich source
of natural bioactive products [4,5]. Marine cyanobacteria in
particular are major producers of various secondary metabolites
with unique structures and biological activities [6,7], including
numerous linear lipopeptides. For example, minnamide A [8],
biseokeaniamides [9], and ikoamide [10], which are all derived
from marine cyanobacteria, show growth-inhibitory activity
2. Results and discussion
The marine cyanobacterium Caldora penicillata (334.4 g, wet
weight) was collected from the coast of Odo, Okinawa, Japan, and
extracted with MeOH. The extract was subjected to fractionation
guided by glucose uptake activity in L6 myotubes with solvent
partitioning, ODS silica gel column chromatography (MeOHeH2O),
and reversed-phase HPLC to yield komesuamide (1) and odopeni-
cillatamide (2).
* Corresponding author. Graduate School of Engineering and Science, University
of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, 903-0213, Japan.
E-mail address: t-teruya@edu.u-ryukyu.ac.jp (T. Teruya).
Komesuamide (1) was obtained as a colorless oil, and its mo-
lecular formula was determined by HRESIMS to be C57H84N8O10
.
https://doi.org/10.1016/j.tet.2021.131969
0040-4020/© 2021 Elsevier Ltd. All rights reserved.

K. Ozaki, A. Jinno, N. Natsume et al.
Tetrahedron 85 (2021) 131969
Fig. 2. Key 2D NMR correlations for 1.
presence of three prolines (Pro1, Pro2, and Pro3), two N-methyl-
phenylalanines (NeMe-Phe1 and NeMe-Phe2), glycine (Gly), N-
methyl-isoleucine (NeMe-Ile), valine (Val), and 2-methylhexanoic
acid (Mha).
The sequence for 1 was determined by HMBC and NOESY ana-
lyses (see Fig. 1). The HMBC signals H-16 (dH 2.95)/C-17 (dC 167.7),
H2-18 (dH 4.14, 3.74)/C-19 (dC 170.4), H-25 (dH 3.13)/C-26 (dC 173.3),
NH (2) (dH 7.28)/C-31 (dC 170.5), and H-40 (dH 2.86)/C-41 (dC 173.2)
and the NOESY correlations H2-5 (dH 3.37, 3.34)/H-8 (dH 5.57), H2-45
Fig. 1. Structures of komesuamide (1) and odopenicillatamide (2).
The NMR data for 1 are summarized in Table 1. The 1H NMR spectra
suggested the presence of two NH groups (dH 7.28, 6.74), three N-
methylamide groups (dH 3.13, 2.95, 2.86), and one methyl ester
moiety (dH 3.71). Additionally, the 1H and 13C NMR spectra revealed
the presence of several
a-protons (~dH 4.40e5.68) and several
carbonyl carbons (~dC 167.7e175.8). Thus, the 1H and 13C NMR
spectra suggested that 1 was a peptidic compound. Analyses of the
COSY, HSQC, and HMBC spectra recorded in CDCl3 revealed the
(dH 3.82, 3.52)/H-47 (dH 4.62), and H2-50 (dH 3.65, 3.52)/H-57 (dH
1.08) established the linear sequence of komesuamide (1) as Pro1-
N-Me-Phe1-Gly-N-Me-Ile-Val-N-Me-Phe2-Pro2-Pro3-Mha. Thus,
Table 1
NMR data for komesuamide (1) in CDCl3.
Unit
Pro1
No.
dCa, type
dH mult (J in Hz)b
Unit
No.
dCa, type
dH mult (J in Hz)b
1
2
3a
3b
4a
4b
5a
5b
6
7
8
9a
9b
10
11/15
12/14
13
16
17
18a
18b
NH (1)
19
20
21
22
23a
23b
24
25
26
27
28
29
30
172.6, C
59.1, CH
29.0, CH2
NeMe-Phe2
31
32
170.5, C
57.1, CH
34.2, CH2
4.40, dd (8.4, 5.6)
2.15, m
1.85, m
1.93, m
1.80, m
3.37, m
3.34, m
3.71, s
5.68, dd (11.8, 5.0)
3.49, m
2.81, m
33a
33b
34
35/39
36/38
37
40
41
42
43a
43b
44a
44b
45a
45b
46
25.2, CH2
47.0, CH2
137.6, C
129.0, CH
128.44c, CH
126.6, CH
31.1, CH3
173.2, C
7.17, m
7.22, m
7.17, m
2.86, s
52.3, CH3
168.1, C
56.2, CH
35.1, CH2
NeMe-Phe1
Pro2
5.57, dd (8.2, 7.2)
3.27, dd (13.7, 8.2)
2,80, dd (13.7, 7.2)
56.6, CH
27.9, CH2
4.64, dd (7.8, 5.5)
1.74, m
1.04, m
1.82, m
1.78, m
137.6, C
25.0, CH2
47.1, CH2
129.6, CH
128.37cCH
126.8, CH
29.8, CH3
167.7, C
7.22, m
7.24, m
7.22, m
2.95, s
3.82, m
3.52, m
Pro3
Mha
170.8, C
57.6, CH
28.3, CH2
Gly
47
4.62, dd (8.2, 4.2)
2.07, m
2.00, m
2.15, m
3.65, m
41.0, CH2
4.14, dd (17.4, 5.1)
3.74, dd (17.4, 3.8)
6.74, brs
48a
48b
49
50a
50b
51
25.1, CH2
47.4, CH2
NeMe-Ile
170.4, C
60.6, CH
31.8, CH
15.7, CH3
24.3, CH2
4.81, d (11.1)
2.12, m
0.91, d (6.6)
1.38, m
1.04, m
0.86, m
3.52, m
175.8, C
37.9, CH
33.5, CH2
52
2.52, m
1.67, m
1.36, m
1.28, m
1.29, m
0.87, m
1.08, d (6.6)
53a
53b
54
55
56
10.6, CH3
31.0, CH3
173.3, C
55.4, CH
30.3, CH
19.4, CH3
18.9, CH3
29.5, CH2
22.9, CH2
14.3, CH3
17.0, CH3
3.13, s
Val
4.56, dd (8.4, 8.3)
2.14, m
0.86, d (6.3)
0.94, d (6.7)
7.28, d (8.3)
57
NH (2)
a
Measured at 125 MHz.
Recorded at 500 MHz.
These signals are interchangeable.
b
c
2

K. Ozaki, A. Jinno, N. Natsume et al.
Tetrahedron 85 (2021) 131969
the gross structure of 1 was determined and is shown in Fig. 2. The
sequence of residues in 1 was further confirmed by analysis of ESI-
MS/MS fragmentation patterns (see Supplementary data Figs. S16 e
S18).
The configurations of the Pro1, NeMe-Phe1, NeMe-Ile, Val,
NeMe-Phe2, Pro2, and Pro3 units in 1 were L, D, L, L, D, L, and L,
respectively, as determined by the acid hydrolysis of 1 followed by
Marfey’s method [13]. To determine the absolute configuration at
C-52 in Mha, we performed the acid hydrolysis of 1 followed by
purification by chromatography on silica gel, derivatization with
phenacyl bromide, and further purification by reversed-phase
Scheme 2. Preparation of phenacyl esters of 2S-Mha (5).
correlations between H-4 and C-13 were not observed. However,
the remaining carbonyl carbon chemical shift (dC 171.7) indicated
the presence of an amide functionality. Also, the linkage of Bn-O-
Me-pyrrolinone and Pro1 was confirmed by its molecular formula
and degree of unsaturation. These data established the linear
sequence of odopenicillatamide (2) as Bn-O-Me-pyrrolinone-Pro1-
N-Me-Val-Pro2-Phe-3-methoxyhexanoic acid. Thus, the gross
structure of 2 was determined and is shown in Fig. 3. The sequence
of residues in 2 was further confirmed by analysis of ESI-MS/MS
fragmentation patterns (see Supplementary data Figs. S29 e S31).
The configurations of the Pro1, NeMe-Val, Pro2, and Phe units in
2 were L, L, L, and D, respectively, as determined by acid hydrolysis
of 2 followed by Marfey’s method. Determination of the configu-
ration at C-4 required ozonolysis followed by oxidative workup,
acid hydrolysis, and derivatization with Marfey’s reagent. HPLC
analysis of this product revealed that the configuration of Phe from
Bn-O-Me-pyrrolinone was L. Therefore, the configuration of C-4
was S. To determine the absolute configuration of C-40, we per-
formed acid hydrolysis followed by derivatization with phenacyl
bromide and then purification by reversed-phase HPLC. This yiel-
ded compound 6 from hydrolysate of 2 (Scheme 3). Next, we syn-
HPLC. This yielded compound
3 from the hydrolysate of 1
(Scheme 1). Next, we synthesized (2S)-Mha (4) according to a
previous report [14] (Scheme 2) and compared the chiral HPLC
retention times of its phenacyl ester (5) and the phenacyl esters of
racemic Mha, which we purchased. This allowed us to assign chiral
HPLC retention times to the phenacyl esters of (2S)-Mha and (2R)-
Mha, respectively. The configuration of C-52 was S as determined
by chiral HPLC analysis of the phenacyl esters. This completed the
structural assignments of komesuamide as shown in 1.
Komesuamide (1) is structurally similar to linear lipopeptides
isolated from cyanobacteria, such as mabuniamide, [12] amanta-
mide [15] and malevamide A [16]. Among the cyanobacterial lip-
opeptides, malevamide A and palau’ imide [17] contain Mha, the
specific fatty acid moiety found in komesuamide (1). In particular,
malevamide A is a close analog of komesuamide (1), with slight
differences in the amino acids and their configurations.
Odopenicillatamide (2) was obtained as a colorless oil, and its
molecular formula was determined by HRESIMS to be C44H59N5O8.
The NMR data for 2 are summarized in Table 2. The 1H NMR spectra
suggested the presence of one NH moiety (dH 6.97), one N-meth-
ylamide moiety (dH 3.03), and two methoxy groups (dH 3.24, 2.75).
thesized
(S)-3-methoxyhexanoic
acid
(9a)
and
(R)-3-
methoxyhexanoic acid (9b), as shown in Scheme 4. The known
compounds 7a and 7b [18] were treated with Meerwein’s reagent
to give compounds 8a and 8b, respectively. Then, removal of the
auxiliary by hydrolysis yielded 9a and 9b, respectively. The
configuration of C-40 was R as determined by chiral HPLC analyses
of phenacyl esters of 6, 10a, and 10b. This completed the structural
assignments of odopenicillatamide as shown in 2.
Odopenicillatamide (2) has linear peptides including Bn-O- Me-
pyrrolinone, and it is therefore related to dolastatin 15 [19] and its
structural analogs such as belamide A [20] and caldoramide [21].
The analogs of dolastatin 15 contain N,N-dimethylvaline moiety,
while 2 contains 3-methoxyhexanoic acid. Among the cyano-
bacterial lipopeptides, micromide [22] contains 3-methoxy hex-
anoic acid moiety, the same fatty acid moiety found in
odopenicillatamide (2).
The 1H and 13C NMR spectra revealed the presence of several
a-
protons (~dH 4.52e5.94) and several carbonyl carbons (~dC
169.3e172.5). Thus, the 1H and 13C NMR spectra suggested that 2
was a peptidic compound. Analyses of the COSY, HSQC, and HMBC
spectra recorded in C6D6 revealed the presence of two prolines
(Pro1, Pro2), N-methyl-valine (NeMe-Val), phenylalanine (Phe),
and 3-methoxyhexanoic acid. The remarkable chemical shifts
recorded for a methine carbon (dC 94.9; dH 4.28) adjacent to the
nonprotonated carbon (dC 177.8) suggested oxygenation at the
latter position. Moreover, HMBC correlations between H-2 (dH 4.28)
and C-1 (dC 169.3); H-2 (dH 4.28) and C-4 (dC 59.8); H-4 (dH 4.63) and
C-2 (dC 94.9); and H2-5 (dH 3.73, 2.89) and C-3 (dC 177.8) were
observed, and these data established a substituted pyrrolinone ring.
The HMBC correlations from H2-5 (dH 3.73, 2.89) to C-6 (dC 135.2)
and C-7/11 (dC 130.8) connected the benzyl moiety to the C-4 po-
sition. Additional HMBC correlations from H-12 (dH 2.75) to C-3 (dC
177.8) connected the methoxy group to the C-3 position. These data
revealed the presence of a 3-methoxy-4-benzylpyrrolinone ring
(Bn-O-Me-pyrrolinone). The sequence for 2 was determined by
HMBC analyses. The HMBC signals H-14 (dH 5.94)/C-18 (dC 169.4),
H-23 (dH 3.03)/C-24 (dC 172.5), H-25 (dH 4.52)/C-29 (dC 169.6), and
NH (dH 6.97)/C-38 (dC 170.2) led to the assignment of the partial
structure of C-13 to C-44. The sequence of Bn-O-Me-pyrrolinone
and Pro1 was not determined using HMBC data because HMBC
The biological activities of komesuamide (1) and odopenicilla-
tamide (2) were evaluated using a glucose uptake assay in cultured
rat L6 myotubes. Both 1 and 2 showed no cytotoxicity at 10e40 mM
(Fig. 4a) and stimulated glucose uptake in a dose-dependent
manner (Fig. 4b). In addition, 1 and 2 also stimulated glucose up-
take in L6 myotubes without insulin.
3. Conclusion
In conclusion, the linear lipopeptides komesuamide (1) and
odopenicillatamide (2) were isolated from the marine cyanobac-
terium Caldora penicillata collected in Okinawa. The structures of
these compounds were established by spectroscopic analyses and
HPLC analyses of the acid hydrolysates. Both compounds stimu-
lated glucose uptake in a dose-dependent in cultured L6 myotubes.
Therefore, this study revealed that marine cyanobacteria can be a
source of linear lipopeptides with glucose uptake regulatory
properties.
Scheme 1. Acid hydrolysis of 1 and preparation of 3.
3

K. Ozaki, A. Jinno, N. Natsume et al.
Tetrahedron 85 (2021) 131969
Table 2
NMR data for odopenicillatamide (2) in C6D6.
Unit
No.
dCa, type
dH mult (J in Hz)b
Unit
Pro2
No.
dCa, type
dH mult (J in Hz)b
Bn-O-Me-pyrrolinone
1
2
3
4
5a
5b
6
7/11
8/10
9
12
13
14
15a
15b
16a
16b
17a
17b
18
19
20
21
22
23
169.3, C
94.9, CH
177.8, C
59.8, CH
35.0, CH2
24
25
26
27a
27b
28a
28b
29
172.5, C
57.1, CH
28.7, CH2
24.5, CH2
4.28, s
4.52, t (6.1)
1.35, m
1.62, m
1.01, m
3.52, m
2.70, m
4.63, brt (3.7)
3.73, dd (14.1, 3.7)
2.89, dd (14.1, 3.7)
47.2, CH2
135.2, C
130.8, CH
128.4, CH
127.2 CH
57.3, CH3
171.7, C
7.51, d (7.5)
7.27, m
7.07, m
Phe
169.6, C
52.6, CH
39.9, CH2
30
5.32, m
3.22, m
3.17, m
31a
31b
32
33/37
34/36
35
NH
38
39a
39b
40
41a
41b
42
43
44
2.75, s
Pro1
138.0, C
60.9, CH
29.3, CH2
5.94, dd (8.6, 4.2)
2.19, m
1.70, m
1.64, m
1.43, m
130.1, CH
128.5, CH
126.9, CH
7.31, m
7.11, m
7.02, m
6.97, d (8.2)
24.6, CH2
48.1, CH2
3-methoxyhexanoic acid
170.2, C
41.1, CH2
3.95, m
3.71, m
2.22, dd (14.7, 7.0)
2.09, dd (14.7, 4.5)
3.51, m
1.42, m
1.32, m
1.28, m
0.83, t (7.2)
3.24, s
NeMe-Val
169.4, C
59.9, CH
28.1, CH
19.4, CH3
19.7, CH3
30.3, CH3
78.1, CH
36.3, CH2
5.36, d (11.3)
2.50, m
1.15, d (6.7)
1.28, d (6.5)
3.03, s
18.7, CH2
14.4, CH3
56.8, CH3
a
Measured at 125 MHz.
Recorded at 500 MHz.
b
thin-layer chromatography (TLC), and spots were visualized with
UV detection (254 nm) or through staining with phosphomolybdic
acid. TLC analysis was conducted on E. Merck precoated silica gel 60
F254. Chromatographic purification of products was performed
using Silica Gel 60 (Nacalai Tesque). Anhydrous CH2Cl2 was used as
obtained from commercial supplies. All moisture-sensitive re-
actions were performed with standard syringe in septa techniques,
and starting materials were azeotropically dried with toluene
before use. Chemicals and solvents were the best grade available
and used as received from commercial sources. The absorbance of
the assay mixture was determined using a BioTek ELx 800 absor-
bance microplate reader.
Fig. 3. Key 2D NMR correlations for 2.
4.2. Identification of the marine cyanobacterium
A cyanobacterial filament was isolated under a microscope and
crushed with freezing and thawing. The 16S rRNA genes were PCR-
amplified in two stages by using two cyanobacterial primer sets:
CYA106F, a cyanobacterial-specific primer, and 16S1541R, a uni-
versal primer for the first amplification, and CYA359F and 16S1371R
Scheme 3. Acid hydrolysis of 2 and preparation of 6.
for the second amplification. The first PCR reaction contained 10
of DNA template, 0.5 L of KOD-Multi & Epi- (TOYOBO, Osaka,
Japan), 12.5
L of 2 ꢀ PCR buffer for KOD-Multi & Epi- (TOYOBO,
Osaka, Japan), 0.5 L of CYA106F and 16S1541R (50 pM, respec-
tively). The PCR reaction was performed as follows: initial dena-
turation for 2 min at 94 ꢁC, amplification by 40 cycles of 10 s at
98 ꢁC, 10 s at 58 ꢁC, 1 min at 68 ꢁC, and final elongation for 7 min at
68 ꢁC. The obtained PCR products were diluted 100-fold with
mL
m
4. Experimental section
m
m
4.1. General experimental procedures
The optical rotation was measured on a JASCO P-1010 polarim-
eter or DIP-1000. UV spectra were measured on a JASCO V-660 UV
visible spectrometer. IR spectra were measured on a JASCO FT/IR-
6100. All NMR data were recorded on a Bruker AVANCE III 500
for 1H (500 MHz) and 13C (125 MHz). Chemical shifts were reported
relative to the residual solvent signals (CDCl3: dH 7.26, dC 77.2, C6D6:
dH 7.16, dC 128.1). ESIMS and ESI-MS/MS data were obtained using a
Waters Quattro micro API mass spectrometer. HRESIMS was per-
formed on a Waters Micromass Q-TOF. HPLC was carried out with a
JASCO PU-2080 Plus Intelligent HPLC pump and a JASCO UV-2075
Plus Intelligent UV/VIS detector. Reactions were monitored by
sterilized water. The second PCR reaction contained 10
diluted PCR product, 0.5 L of KOD-Multi & Epi- (TOYOBO, Osaka,
Japan), 12.5
L of 2 ꢀ PCR buffer for KOD-Multi & Epi- (TOYOBO,
Osaka, Japan), 0.5 L of CYA359F and 16S1371R (50 pM, respec-
mL of the
m
m
m
tively). The second PCR reaction was performed as follows: initial
denaturation for 2 min at 94 ꢁC, amplification by 40 cycles of 10 s at
98 ꢁC and 90 s at 66 ꢁC. PCR products were analyzed on agarose gel
(1%) in TBE buffer and visualized by ethidium bromide staining. The
obtained DNA was sequenced with CYA359F and 16S1371R primers.
4

K. Ozaki, A. Jinno, N. Natsume et al.
Tetrahedron 85 (2021) 131969
Scheme 4. Synthesis of authentic 3-methoxyhexanoic acid.
with default settings was used to select the best model of DNA
substitution for the Maximum Likelihood (ML) analysis and
Bayesian analysis according to the Akaike information criterion
(AIC). The ML analysis was conducted by PhyML (version 20131016)
[27], using the TIM2þI þ G model with a gamma shape parameter
of 0.4590, a proportion of invariant sites of 0.4820 and nucleotide
frequencies of F(A) ¼ 0.2449, F(C) ¼ 0.2278, F(G) ¼ 0.3174,
F(T) ¼ 0.2099. Bootstrap resampling was performed on 1000 rep-
licates. The ML tree was visualized with Njplot (version 2.3) [28].
The Bayesian analysis was conducted by MrBayes (version 3.2.5)
[29] using the GTR þ I þ G model. The Markov chain Monte Carlo
process was set at 2 chains, and 1,000,000 generations were con-
ducted. Sampling frequency was assigned at every 500 generations.
After analysis, the first 100,000 trees were deleted as burn-in, and
the consensus tree was constructed. The Bayesian tree was visu-
alized with FigTree (version 1.4.0, http://tree.bio.ed.ac.uk/software/
figtree). As a result, the cyanobacterium (accession no. LC590897)
formed a clade with Caldora penicillata. Therefore, the cyanobac-
terium was classified into Caldora penicillata.
The marine cyanobacterium was morphologically classified into
the genus Caldora penicillata [23], because the cells were red cy-
lindrical shape with 5.2 0.6
m
m wide (n ¼ 10) and 6.5 1.0
mm
long (n ¼ 10), trichomes lacked heterocysts or other specialized
Fig. 4. Effects of 1 and 2 on cell viability and glucose uptake in cultured L6 myotubes.
(a) Cells were treated with the indicated concentrations of compounds. After incuba-
tion for 20 h, cell viability was determined based on an MTT assay. Values are the
cells, and terminal cells of filaments were rounded.
mean
SD of triplicate determinations. (b) Cells were preincubated in Krebs-
4.3. Extraction and isolation
Henseleit-HEPES buffer (KHH buffer) without glucose for 2 h. They were then incu-
bated in KHH buffer containing 11 mM glucose with the incubated concentrations of
compounds for 20 h. Glucose uptake was measured using a Glucose C-II Test kit.
The marine cyanobacterium Caldora penicillata (334.4 g, wet
weight), was collected at Odo, Okinawa, Japan (26ꢁ090N, 127ꢁ710E),
in July 2017. The cyanobacterium was extracted with MeOH (1.8 L)
at room temperature (rt) for 40 days. The extract was filtered, and
the filtrate was concentrated. The residue was partitioned between
H2O (0.2 L) and EtOAc (0.2 L ꢀ 3). The material obtained from the
organic layer was further partitioned between 90% aqueous MeOH
(0.1 L) and n-hexane (0.1 L ꢀ 3). The aqueous 90% MeOH fraction
(0.29 g) was separated by column chromatography on ODS (5.5 g)
using 60% aqueous MeOH, 80% aqueous MeOH, and MeOH. The
fraction (82.3 mg) that eluted with 80% aqueous MeOH was sub-
Mabuniamide [12] (Mab) was used as a positive control. Values are mean
triplicate determinations.
SD of
This sequence is available in the DDBJ/EMBL/Genbank databases
under an accession number LC590897. The nucleotide sequence of
16S rRNA gene obtained in this study was used for phylogenetic
analysis with the sequences of related cyanobacterial 16S rRNA
genes [23]. All sequences were aligned by SINA web service
(version 1.2.11) [24] with default settings. The poorly aligned po-
sitions and divergent regions were removed by Gblocks Server
(version 0.91b) [25], implementing the options for a less stringent
selection, including the ‘Allow smaller final blocks’, ‘Allow gap
positions within the final blocks’ and ‘Allow less strict flanking
positions’ options. The obtained 893 nucleotide positions have
been used for phylogenetic analysis. JModeltest (version 2.1.7) [26]
jected to reversed-phase HPLC [Cosmosil 5C18-AR-II
(f
20 ꢀ 250 mm), 80% MeOH at 5.0 mL/min, UV detection at 215 nm]
to give seven fractions. Fraction four (tR ¼ 48.2 min) was subjected
to further HPLC [Cosmosil 5C18-AR-II (
at 4.0 mL/min, UV detection at 215 nm] to yield komesuamide (1,
10.2 mg, tR ¼ 12.1 min). Furthermore, fraction two (tR ¼ 36.2 min)
f
10 ꢀ 250 mm), 60% MeCN
5

K. Ozaki, A. Jinno, N. Natsume et al.
Tetrahedron 85 (2021) 131969
was subjected to further HPLC [Cosmosil 5C18-AR-II
(
f
above. Compound 3 was subjected to chiral HPLC [DAICEL CHIR-
10 ꢀ 250 mm), 60% MeCN at 4.0 mL/min, UV detection at 215 nm] to
ALPAK IC (
f
4.6 ꢀ 250 mm), n-hexane/EtOH ¼ 99:1 at 1.0 mL/min,
yield odopenicillatamide (2, 14.1 mg, tR ¼ 13.0 min). The purity of 1
UV detection at 254 nm]. The retention times (min) of phenacyl
esters of the authentic standards were as follows: (2S)-Mha (5)
(11.2) and racemic mixture (10.4, 11.2). The retention times of
phenacyl esters of (2R)-Mha was assigned to 10.4 min. The reten-
tion time of 3 was 11.2 min.
and 2 were determined as >95% by HPLC analysis.
27
Komesuamide (1): colorless oil; [
a]
ꢂ33.2 (c 1.00, MeOH); UV
D
(MeOH) lmax (logε) 231 (3.88) nm; IR (neat) 3489, 3397, 3322, 3061,
3028, 2961, 2933, 2873, 1746, 1645, 1436, 1272, 1201, 1174, 1110,
1032, 749, 701 cmꢂ1; 1H NMR and 13C NMR data, Table 1; HRESIMS
m/z 1063.6191 [M þ Na]þ (calcd for C57H84N8O10Na, 1063.6191).
4.7. Acid hydrolysis of 2
Odopenicillatamide (2): colorless oil; [
a
]
D
27 ꢂ20.9 (c 1.00, MeOH);
UV (MeOH) lmax (logε) 241 (5.03) nm; IR (neat) 3303, 2960, 2936,
Odopenicillatamide (2, 0.7 mg) was treated with 6 M HCl
2873, 1726, 1693, 1633, 1445, 1376, 1311, 1257, 1198, 1087, 971, 808,
(500
to dryness and subjected to HPLC [Cosmosil HILIC
m
L) at 110 ꢁC for 20 h. The hydrolyzed product was evaporated
702 cmꢂ1
;
1H NMR and 13C NMR data, Table 2; HRESIMS m/z
(f
808.4261 [M þ Na]þ (calcd for C44H59N5O8Na, 808.4261).
4.6 ꢀ 250 mm), MeCN/10 mM AcONH4 ¼ 85:15 at 1.0 mL/min, UV
detection at 215 nm] to yield Pro, NeMe-Val and Phe. Each amino
4.4. Acid hydrolysis of 1
acid was mixed with 0.1% solution of L-FDAA (200
and 0.5 M NaHCO3 (200
L) followed by heating at 40 ꢁC for 90 min.
After cooling to rt, the reaction mixture was neutralized with 2 M
HCl (50 L) and diluted with MeOH (500 L). The solution was
subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II
mL) in acetone
m
Komesuamide (1, 2.0 mg) was treated with 1,4-dioxane (200 mL)
and 6 M HCl (200
m
L) at 110 ꢁC for 19 h. The hydrolyzed product was
m
m
evaporated to dryness and subjected to HPLC [Cosmosil 5C18-PAQ
(f
(
f
4.6 ꢀ 250 mm), H2O with 0.1% trifluoroacetic acid (TFA) at
4.6 ꢀ 250 mm), MeOH/20 mM AcONa ¼ 50:50 (solvent C) or 45:55
(solvent D) at 1.0 mL/min, UV detection at 340 nm]. The L-DAA
derivatives of standard amino acids were prepared by the same
procedure. The retention times (min) of the derivatives of authentic
standards were as follows: NeMe-L-Val (9.2), NeMe-D-Val (19.4),
L-Phe (9.9) and D-Phe (26.0) in solvent C, L-Pro (6.0) and D-Pro (9.7)
in solvent D. The retention time and ESIMS product ion (m/z [M þ
Na]þ) of the L-DAA derivative of NeMe-Val and Phe from hydro-
lysate were 9.2 min (406.1) and 26.0 min (440.0) in solvent C,
respectively. The retention times and ESIMS product ions (m/z [M þ
Na]þ) of the L-DAA derivatives of Pro from hydrolysate was 6.0 min
(390.2) in solvent D.
1.0 mL/min, UV detection at 215 nm] to yield Pro, NeMe-Phe,
NeMe-Ile and Val. Each amino acid was mixed with 0.1% solution of
N
a
-(6-fluoro-2,4-dinitrophenyl)-
reagent, 100 L) in acetone and 0.5 M NaHCO3 (100
heating at 80 ꢁC for 10 min. After cooling to rt, the reaction mixture
was neutralized with 2 M HCl (25 L) and diluted with MeOH
(500 L). The solution was subjected to reversed-phase HPLC
[Cosmosil 5C18-AR-II 4.6 250 mm), MeOH/20 mM
L-alaninamide (L-FDAA, Marfey’s
m
mL) followed by
m
m
(f
ꢀ
AcONa ¼ 60:40 (solvent A), 55:45 (solvent B), 50:50 (solvent C) or
45:55 (solvent D) at 1.0 mL/min, UV detection at 340 nm]. The L-
DAA derivatives of standard amino acids were prepared by the
same procedure. The retention times (min) of the derivatives of
authentic standards were as follows: NeMe-L-Ile (6.7), NeMe-L-
allo-Ile (7.0), NeMe-D-Ile (13.4) and NeMe-D-allo-Ile (12.3) in
solvent A, NeMe-L-Phe (7.2) and NeMe-D-Phe (9.4) in solvent B, L-
Val (7.2) and D-Val (20.4) in solvent C, L-Pro (5.7) and D-Pro (9.4) in
solvent D. The retention time and ESIMS product ion (m/z [M þ
Na]þ) of the L-DAA derivative of NeMe-Ile from hydrolysate was
6.7 min (420.1) in solvent A. The retention time and ESIMS product
ion (m/z [M þ Na]þ) of the L-DAA derivative of NeMe-Phe from
hydrolysate was 9.4 min (454.3) in solvent B. The retention time
and ESIMS product ion (m/z [M þ Na]þ) of the L-DAA derivative of
Val from hydrolysate was 7.2 min (392.2) in solvent C. The retention
time and ESIMS product ion (m/z [M þ Na]þ) of the L-DAA deriva-
tive of Pro from hydrolysate was 5.7 min (390.0) in solvent D.
4.8. Ozonolysis and acid hydrolysis of 2
Ozone gas was bubbled through a solution of odopenicillata-
mide (2, 1.0 mg) in MeOH (2.0 mL) at 0 ꢁC for 2 min. The solution
was then added to a stream of nitrogen gas to remove ozone and
warmed to rt. The solution was oxidized by addition of 30% H2O2
(3.0 mL) and stirred for 1 h. The mixture was concentrated, and the
residue was treated with 6 M HCl (500
hydrolyzed product was evaporated to dryness and subjected to
HPLC [Cosmosil HILIC 4.6 250 mm), MeCN/10 mM
AcONH4 ¼ 85:15 at 1.0 mL/min, UV detection at 215 nm] to yield
Phe. Phe was mixed with 0.1% solution of L-FDAA (200 L) in
acetone and 0.5 M NaHCO3 (200
L) followed by heating at 40 ꢁC for
90 min. After cooling to rt, the reaction mixture was neutralized
with 2 M HCl (50 L) and diluted with MeOH (500 L). The solution
was subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II (
m
L) at 110 ꢁC for 22 h. The
(
f
ꢀ
m
m
4.5. Preparation of compound 3
m
m
f
Komesuamide (1, 3.0 mg) was dissolved in 1,4-dioxane (200
and 9 M HCl (200
L). The solution was stirred at 80 ꢁC for 3 h. The
reaction mixture was evaporated to dryness. The residue was
treated with 100 L of a phenacyl bromide solution (12 mg/mL in
acetone) and 100 L of a triethylamine solution (10 mg/mL in
acetone) followed by heating at 50 ꢁC for 2 h. The reaction mixture
was evaporated to dryness and subjected to HPLC [Cosmosil 5C18
AR-II (
m
L)
4.6 ꢀ 250 mm), MeOH/20 mM AcONa ¼ 50:50 (solvent C) at 1.0 mL/
min, UV detection at 340 nm]. The retention times (min) of the
derivatives of authentic standards were as follows: L-Phe (9.9) and
D-Phe (26.0) in solvent C. The retention time and ESIMS product ion
(m/z [M þ Na]þ) of the L-DAA derivative of Phe from hydrolysate
was 9.9 min (440.1) in solvent C.
m
m
m
-
f
10 ꢀ 250 mm), 75% MeOH at 5.0 mL/min, UV detection at
4.9. Preparation of compound 6
215 nm] to yield phenacyl ester of 3-methoxyhexanoic acid (3,
0.1 mg, tR ¼ 11.3 min). ESIMS m/z [M þ Na]þ 271.3.
Odopenicillatamide (2, 1.2 mg) was dissolved in 1,4-dioxane
(200
for 3.5 h. The reaction mixture was evaporated to dryness. The
residue was treated with 100 L of a phenacyl bromide solution
(12 mg/mL in acetone) and 100 L of a triethylamine solution
mL) and 9 M HCl (200 m
L). The solution was stirred at 80 ꢁC
4.6. Chiral-phase HPLC analysis of phenacyl esters 3, 5 and racemic
mixture
m
m
Phenacyl esters (5) of authentic 2-methylhexanoic acid (TCI,
Tokyo, Japan) were prepared by the same procedure described
(10 mg/mL in acetone) followed by heating at 50 ꢁC for 2 h. The
reaction mixture was evaporated to dryness and subjected to HPLC
6

K. Ozaki, A. Jinno, N. Natsume et al.
Tetrahedron 85 (2021) 131969
[Cosmosil 5C18-AR-II (
f
10 ꢀ 250 mm), 70% MeOH at 5.0 mL/min,
CDCl3)
d
177.2, 77.6, 57.0, 39.2, 35.9, 18.4, 14.2; HRESIMS m/z
UV detection at 215 nm] to yield phenacyl ester of 3-
methoxyhexanoic acid (6, 0.2 mg, tR ¼ 7.4 min). ESIMS m/z [M þ
Na]þ 287.1.
169.0840 [M þ Na]þ (calcd for C7H14O3Na, 169.0841).
4.11.4. (S)-1-((R)-4-Benzyl-2-thioxothiazolidin-3-yl)-3-
methoxyhexane-1-one (8b)
4.10. Chiral-phase HPLC analysis of phenacyl esters 6, 10a and 10b
To a stirred solution of known compound 7b [18] (28.2 mg,
87.3
molecular sieves (0.498 g). After stirring at rt for 20 min, Proton
Sponge (59.8 mg, 279.0 mol) and Me3OBF4 (47.7 mg, 322.5 mol)
mmol) in anhydrous CH2Cl2 (800 mL) was added dried 4 Å
Phenacyl esters of authentic 3-methoxyhexanoic acids (10a,
10b) were prepared by the same procedure described above.
m
m
Compound 6 was subjected to chiral HPLC [DAICEL CHIRALPAK IC (
f
were added to the mixture. After the mixture was stirred for
20 min, the suspension was diluted with CH2Cl2 and filtered
through a cotton plug. The solid was washed with more CH2Cl2, and
the combined filtrate and washings were evaporated. The residual
oil was purified by column chromatography on silica gel (hexane-
EtOAc, 10:1 v/v) to give three fractions. Furthermore, fraction two
4.6 ꢀ 250 mm), n-hexane/EtOH ¼ 96:4 at 1.0 mL/min, UV detection
at 254 nm]. The retention times (min) of the authentic standards
were as follows: (S)-3-methoxyhexanoic acid (10a) (13.8) and (R)-
3-methoxyhexanoic acid (10b) (12.2). The retention time of 6 was
12.2 min.
was subjected to HPLC [Cosmosil 5C18-AR-II (
MeOH at 5.0 mL/min, UV detection at 215 nm] to yield 8b (3.4 mg,
f
10 ꢀ 250 mm), 80%
4.11. Synthesis of fatty acids
10.1
m
mol, 11.5%, tR ¼ 14.4 min) as a bright-yellow oil; [
a]
26 þ205 (c
D
4.11.1. (2S)-2-methylhexanoic acid (4)
0.78, CH2Cl2); IR (neat) 2957, 2930, 2872, 1698, 1455, 1342, 1262,
Compound 4 was synthesized according to a previous report
[14], starting from hexanoic acid and (S)-4-benzyl-2-oxazolidinone.
1164, 1138, 1040, 747, 702 cmꢂ1
;
1H NMR (500 MHz, CDCl3)
d
7.36e7.27 (m, 5H), 5.38e5.32 (m, 1H), 3.82e3.76 (m, 1H), 3.57 (dd,
The 1H and 13C NMR spectrum matched with reported data.
J ¼ 16.8, 6.6 Hz, 1H), 3.43 (dd, J ¼ 16.8, 5.8 Hz, 1H), 3.37 (ddd,
J ¼ 11.4, 7.2, 0.9 Hz, 1H), 3.37 (s, 3H), 3.25 (dd J ¼ 13.1, 3.8 Hz, 1H),
3.04 (dd, J ¼ 13.1, 10.7 Hz, 1H), 2.89 (dd, J ¼ 11.4, 0.6, 1H), 1.58e1.47
(m, 2H), 1.47e1.33 (m, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H); 13C NMR
23
[
a]
¼ þ21.8 (c 0.82, Et2O); HRESIMS m/z 129.0916 [M ꢂ H]- (calcd
D
for C7H13O2, 129.0916).
4.11.2. (S)-1-((S)-4-benzyl-2-thioxothiazolidin-3-yl)-3-
methoxyhexane-1-one (8a)
(125 MHz, CDCl3) d 201.3, 172.4, 136.7, 129.6, 129.1, 127.4, 77.5, 68.9,
57.0, 43.2, 36.9, 36.3, 32.1, 18.6, 14.3; HRESIMS m/z 322.1477 [M þ
To a stirred solution of known compound 7a [18] (30.9 mg,
H]þ (calcd for C17H24NO3S, 322.1477).
95.6
molecular sieves (0.425 g). After stirring at rt for 20 min, Proton
Sponge (73.7 mg, 343.9 mol) and Me3OBF4 (43.6 mg, 294.8 mol)
mmol) in anhydrous CH2Cl2 (800 mL) was added dried 4 Å
4.11.5. (R)-3-methoxyhexanoic acid (9b)
m
m
Compound 8b (10.0 mg, 29.6
mmol) was dissolved in THF
were added to the mixture. After the mixture was stirred for
20 min, the suspension was diluted with CH2Cl2 and filtered
through a cotton plug. The solid was washed with more CH2Cl2, and
the combined filtrate and washings were evaporated. The residual
oil was purified by column chromatography on silica gel (hexane-
EtOAc, 10:1 v/v) to give three fractions. Furthermore, fraction two
(200 L) and aqueous LiOH (1.0 M, 110
mixture was stirred at rt for 80 min. The reaction mixture was
concentrated and dissolved in H2O and acidified with 1 M HCl. The
m
m
L, 110 mol), and the
m
mixture was concentrated and subjected to HPLC [Cosmosil
10 ꢀ 250 mm), 40% MeOH with TFA at 5.0 mL/min, UV detection
at 215 nm] to yield 9b (2.9 mg, 21.8 mol, 73.6%) as a colorless oil;
ꢂ2.63 (c 0.18, CHCl3); IR (neat) 2961, 2935, 2875, 1713, 1411,
pNAP
(f
m
27
was subjected to HPLC [Cosmosil 5C18-AR-II (
MeOH at 5.0 mL/min, UV detection at 215 nm] to yield 8a (2.7 mg,
f
10 ꢀ 250 mm), 80%
[a]
D
1175, 1092 cmꢂ1 1H NMR (500 MHz, CDCl3)
; d 3.67e3.61 (m, 1H),
26
8.0
0.78, CH2Cl2); IR (neat) 2957, 2930, 2871, 1698, 1455, 1342, 1264,
1164, 1138, 1042, 748, 702 cmꢂ1 1H NMR (500 MHz, CDCl3)
7.37e7.27 (m, 5H), 5.38e5.32 (m,1H), 3.87e3.82 (m, 1H), 3.49 (dd,
mmol, 8.4%, tR ¼ 16.8 min) as a bright-yellow oil; [
a]
þ205 (c
3.40 (s, 3H), 2.54 (d, J ¼ 6.7 Hz, 1H), 2.53 (d, J ¼ 5.3 Hz, 1H),
D
1.65e1.56 (m, 1H), 1.54e1.45 (m, 1H), 1.44e1.32 (m, 2H), 0.94 (t,
;
J ¼ 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 175.3, 77.5, 57.1, 38.9,
d
35.8, 31.1, 18.4, 14.2; HRESIMS m/z 169.0841 [M þ Na]þ (calcd for
C7H14O3Na, 169.0841). The 1H NMR spectrum matched with re-
ported data [30].
J ¼ 17.1, 8.6 Hz, 1H), 3.39 (ddd, J ¼ 11.4, 7.2, 0.9 Hz, 1H), 3.37 (s, 3H),
3.28 (dd, J ¼ 17.1, 3.6 Hz, 1H), 3.25 (dd, J ¼ 13.1, 3.8 Hz, 1H), 3.05 (dd,
J ¼ 13.1, 10.7 Hz, 1H), 2.88 (d, J ¼ 11.4 Hz, 1H), 1.64e1.55 (m, 1H),
1.55e1.48 (m, 1H), 1.43e1.35 (m, 2H), 0.95 (t, J ¼ 7.3 Hz, 3H) Some
signals overlapped with a water signal; 13C NMR (125 MHz, CDCl3)
4.12. Culturing of L6 myotubes
d
201.4, 172.4, 136.7, 129.6, 129.0, 127.4, 77.1, 68.8, 57.3, 43.8, 36.8,
L6 myoblasts were purchased from the Japanese Collection of
Research Bioresources. L6 myotubes cells were cultured in DMEM
containing 10% (v/v) FBS and 2.0% penicillin/streptomycin in a hu-
midified 5% CO2 incubator at 37 ꢁC. To differentiate myotubes, L6
myoblasts were seeded at 5.0 ꢀ 103 cells/well in 96-well plates and
cultured to 90% confluency for 2 days. Then the cells were cultured
to form myotubes in DMEM containing 2% FBS for 1 week. The
medium was renewed every 2 days.
35.8, 32.2, 18.5, 14.4; HRESIMS m/z 344.1294 [M þ Na]þ (calcd for
C
17H23NO3SNa, 344.1296).
4.11.3. (S)-3-methoxyhexanoic acid (9a)
Compound 8a (9.2 mg, 27.3 mol) was dissolved in THF (200
and aqueous lithium hydroxide solution (LiOH) (1.0 M, 110
110 mol), and the mixture was stirred at rt for 50 min. The reaction
m
mL)
mL,
m
mixture was concentrated and dissolved in H2O and acidified with
1 M HCl. The mixture was concentrated and subjected to HPLC
4.13. Determination of glucose uptake
[Cosmosil
p
NAP (
f
10 ꢀ 250 mm), 40% MeOH with 0.1% TFA at
5.0 mL/min, UV detection at 215 nm] to yield 9a (2.8 mg, 21.2
mmol,
L6 myotubes were incubated in filter-sterilized Krebs-Henseleit-
HEPES buffer (1.2 mM MgSO4, 1.2 mM KH2PO4, 4.7 mM KCl, 119 mM
NaCl, 2.5 mM CaCl2, 25 mM NaHCO3, pH 7.4) containing 0.1% bovine
serum albumin, 10 mM HEPES and 2 mM sodium pyruvate (KHH
buffer) for 2 h. The myotubes were then cultured for 22 h in KHH
buffer containing 11 mM glucose without or with komesuamide (1,
77.6%) as a colorless oil; [
a]
D
27 þ2.16 (c 0.25, CHCl3); IR (neat) 2961,
2934, 2875, 1714, 1435, 1200, 1092 cmꢂ1; 1H NMR (500 MHz, CDCl3)
d
3.70e3.63 (m, 1H), 3.39 (s, 3H), 2.61e2.46 (m, 2H), 1.65e1.54 (m,
1H), 1.54e1.44 (m, 1H), 1.44e1.30 (m, 2H), 0.93 (t, J ¼ 7.3 Hz, 3H)
Some signals overlapped with a water signal; 13C NMR (125 MHz,
7

K. Ozaki, A. Jinno, N. Natsume et al.
Tetrahedron 85 (2021) 131969
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mM) and odopenicillatamide (2, 10e40 mM), respectively.
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4.14. Cell viability
After the glucose uptake assay, an MTT solution (10 mL, 5 mg/mL
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in H2O) was added to each well, and the plate was incubated at
37 ꢁC with 5% CO2 for 4 h. All remaining supernatant was then
removed, and DMSO (100 mL) was added to each well to dissolve the
resultant formazan crystals. Absorbance was measured by using a
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Declaration of competing interest
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